Cellulase variants

Abstract

The present invention provides variants of a parent cellulase, such as, e.g., a cellulase classified in family 45 such as a Humicola insolens 43 kD endoglucanase, comprising a cellulose binding domain (CBD), a catalytically active domain (CAD), and a region linking the CBD and CAD (the linking region), wherein one or more amino acid residues of the CBD, CAD, or linking region is deleted or substituted by one or more amino acid residues and/or one or more amino acids are added to the linking region and/or another CBD is added at the opposite end of the CAD. Such variants have improved properties as regards, e.g., alkaline activity, compatibility with detergent composition ingredients, particular soil removal, color clarification, defuzzing, depilling, harshness reduction, and sensitivity to anionic surfactants. Such variants are useful, e.g., in detergent compositions, for textile treatment, in paper pulp processing, for animal feed, and for stone-washing of jeans.

We claim: 
     
       1. A cellulase variant of a microbial parent cellulase having a catalytically active domain classified in family 45, said variant comprising a cellulose binding domain (CBD), a catalytically active domain (CAD) and a region linking the cellulose binding domain and catalytically active domain (the linking region), wherein said variant comprises a substitution of one or more amino acid residues at a position corresponding to a position in SEQ ID NO:10 selected from the group consisting of: (a) positions 4, 5, 6, 7, 8, 10, 11, 12, 13, 15, 18, 20, 21, 44, 45, 48, 74, 82, 90, 110, 114, 117, 119, 121, 128, 131, 132, 147, 176, 178 or 179, wherein the substitution is predicted to alter the surface conformation of the active site cleft;   (b) positions 111, 112, 113, 114, 115, 116, 117, 118 or 119, wherein the substitution is predicted to alter the hydrogen bonding properties of the flexible loop region;   (c) positions 9, 14, 28, 37, 55, 58, 59, 60, 63, 72, 73, 78, 109, 118, 123, 129, 131, 132, 133, 136, 142, 145, 146, 158, 163, 176, 179, 186 or 196, wherein the substitution is predicted to alter the surface conformation of a channel;   (d) positions 2, 13, 20, 44, 65, 66, 67, 90, 95, 96, 100, 102, 103, 175, 176, 178, 180, 183 or 185, wherein the substitution is predicted to alter the positive electrostatic charge of a positively charged surface region;   (e) positions 55, 74, 90 or 123, wherein the substitution is predicted to alter the negative charge of a cleft;   (f) positions 37, 62, 63, 78, 118, 129, 131, 133, 136, 142, 146, 158, 162, 163, 175, 176, 179, 186 or 196, wherein the substitution is predicted to alter the sensitivity of the variant to anionic surfactants;   (g) positions 240 and 241 in the linking region;   (h) positions 251, 252, 253, 254, 255, 261, 262, 265, 268, 269, 274, 275, 276, 277, 278, 279, 280, 281, or 282 in the cellulose binding domain; and   (i) combinations of any of the foregoing.   
     
     
       2. A cellulase variant as defined in claim 1, wherein said substitutions are selected from the group consisting of:   ______________________________________                                    
           D2N                                                            
           S5A                                                            
           T6S                                                            
           W9S,G                                                          
           D10E                                                           
           K13R                                                           
           S15N,A,D                                                       
           W18H                                                           
           K20R                                                           
           V28T                                                           
           R37N,S,A                                                       
           K44R                                                           
           S45N,D,A                                                       
           E48D,Q,A                                                       
           S55E,D                                                         
           D58N,S,A                                                       
           Q59S,A,G                                                       
           N65R                                                           
           D66R,N                                                         
           D67R,N                                                         
           A74D,N,S                                                       
           Y90F                                                           
           S96R                                                           
           A100R                                                          
           K102R                                                          
           K103R                                                          
           S110N,A,D                                                      
           T111G,A,S                                                      
           G112A                                                          
           G113A                                                          
           L115I,V,F,H,T,N,Q.G                                            
           G116A                                                          
           S116G,A,D,E,N,Q                                                
           N118G,A,S,D,R                                                  
           H119Q,K                                                        
           S123D,E,Y                                                      
           K175R                                                          
           N179D,H,A                                                      
           S185R,K                                                        
           C11A + C135A                                                   
           C12A + C47A and                                                
           R37N + D58A.                                                   
______________________________________                                    
     
     
     
       3. A cellulase variant as defined in claim 1, wherein said substitutions are selected from the group consisting of:   ______________________________________                                    
           R37N,S,A                                                       
           A63D,T,R                                                       
           A78D                                                           
           N118D                                                          
           V129D,T,S                                                      
           I131L,V,T,N,Q,H,G                                              
           D133Q                                                          
           T136D                                                          
           L142D,T,S                                                      
           R146E,Q,S                                                      
           R158D                                                          
           L163N                                                          
           N176D                                                          
           N179D                                                          
           N186D                                                          
           R196D                                                          
           A78P                                                           
           A162P and                                                      
           K175G,S.                                                       
______________________________________                                    
     
     
     
       4. A cellulase variant as defined in claim 1, wherein said substitutions are selected from the group consisting of:   ______________________________________                                    
           E251S,Q,N,P                                                    
           V268E                                                          
           A269E,R                                                        
           T265R,E                                                        
           W253Y,F                                                        
           A254S,D,G                                                      
           Q255E,R,K                                                      
           W261R,Y,F                                                      
           S262A,N,D                                                      
           T274R                                                          
           K275R,Q                                                        
           I276D,Q,N                                                      
           N277Q,D                                                        
           D278P                                                          
           W279Y,F                                                        
           H281S                                                          
           Q282N,R and                                                    
           Y280F + Q282N.                                                 
______________________________________                                    
     
     
     
       5. A cellulase variant as defined in claim 1, wherein the parent cellulase is a fungal cellulase. 
     
     
       6. A cellulase variant as defined in claim 5, wherein the parent cellulase is derived from a strain of Humicola, Trichoderma, Myceliophthora, Penicillium, Irpex, Aspergillus or Fusarium. 
     
     
       7. A detergent composition comprising a cellulase variant as defined in claim 6. 
     
     
       8. A detergent composition as defined in claim 7, further comprising one or more enzymes selected from the group consisting of protease, lipase, peroxidase, esterase, cellulase, endoglucanase type II, oxidase and amylase. 
     
     
       9. A detergent composition as defined in claim 7, wherein the detergent composition is a powder or a liquid composition. 
     
     
       10. A detergent composition as defined in claim 8, wherein said powder composition is a compact heavy duty powder composition and said liquid composition is a heavy duty liquid composition or a compact heavy duty liquid composition. 
     
     
       11. A cellulase variant of a microbial parent cellulase having a catalytically active domain classified in family 45, said variant comprising a cellulose binding domain (CBD), a catalytically active domain (CAD) and a region linking the cellulose binding domain and catalytically active domain (the linking region), wherein one or more amino acid residues on the surface of the CAD, the CBD, or the linking region are substituted by one or more amino acid residues which are less sensitive to oxidation or the presence of a peroxidase bleaching system and   wherein said substitution is predicted to alter the sensitivity of the cellulase variant to oxidation or to the presence of bleaching agents.   
     
     
       12. A cellulase variant as defined in claim 11, wherein one or more methionine, tryptophan or tyrosine are substituted by serine, asparagine, glutamine, proline, phenylalanine, glutamic acid, arginine or glycine. 
     
     
       13. A cellulase variant as defined in claim 11, wherein said one or more amino acid residues are at a position corresponding to a position in SEQ ID NO:10 selected from the group consisting of 8, 9, 18, 62, 104 and 147. 
     
     
       14. A cellulase variant as defined in claim 13, wherein said substitutions are selected from the group consisting of:   ______________________________________                                    
           W9F,H,S,A;                                                     
           W18H,F,A; and                                                  
           M104S,N,Q.                                                     
______________________________________                                    
     
     
     
       15. A cellulase variant as defined in claim 11, wherein the parent cellulase is a fungal cellulase. 
     
     
       16. A cellulase variant as defined in claim 15, wherein the parent cellulase is derived from a strain of Humicola, Trichoderma, Myceliophthora, Penicillium, Irpex, Aspergillus or Fusarium. 
     
     
       17. A detergent composition comprising a cellulase variant as defined in claim 11. 
     
     
       18. A detergent composition as defined in claim 17, further comprising one or more enzymes selected from the group consisting of protease, lipase, peroxidase, esterase, cellulase, endoglucanase type II, oxidase and amylase. 
     
     
       19. A detergent composition as defined in claim 17, wherein the detergent composition is a powder or a liquid composition. 
     
     
       20. A detergent composition as defined in claim 19, wherein said powder composition is a compact heavy duty powder composition and said liquid composition is a heavy duty liquid composition or a compact heavy duty liquid composition.